Bcl-2 family inhibitors sensitize human cancer models to therapy

BH3 mimetics, targeting the Bcl-2 family anti-apoptotic proteins, represent a promising therapeutic opportunity in cancers. ABT-199, the first specific Bcl-2 inhibitor, was approved by FDA for the treatment of several hematological malignancies. We have recently discovered IS21, a novel pan BH3 mimetic with preclinical antitumor activity in several tumor types. Here, we evaluated the efficacy of IS21 and other BH3 mimetics, both as single agents and combined with the currently used antineoplastic agents in T-cell acute lymphoblastic leukemia, ovarian cancer, and melanoma. IS21 was found to be active in T-cell acute lymphoblastic leukemia, melanoma, lung, pancreatic, and ovarian cancer cell lines. Bcl-xL and Mcl-1 protein levels predicted IS21 sensitivity in melanoma and ovarian cancer, respectively. Exploring IS21 mechanism of action, we found that IS21 activity depends on the presence of BAX and BAK proteins: complexes between Bcl-2 and Bcl-xL proteins and their main binding partners were reduced after IS21 treatment. In combination experiments, BH3 mimetics sensitized leukemia cells to chemotherapy, ovarian cancer cells and melanoma models to PARP and MAPK inhibitors, respectively. We showed that this enhancing effect was related to the potentiation of the apoptotic pathway, both in hematologic and solid tumors. In conclusion, our data suggest the use of inhibitors of anti-apoptotic proteins as a therapeutic strategy to enhance the efficacy of anticancer treatment.


INTRODUCTION
Cancer patients often show intrinsic/innate or acquired resistance to treatments that ultimately result in death [1,2]; thus, managing these patients represents a challenge, and new therapeutic options are needed. One attractive strategy is the combination of currently available therapies with inhibitors of anti-apoptotic proteins from the Bcl-2 family, which are often expressed at high levels in various types of cancers [3,4]. Through the interaction with pro-apoptotic proteins, anti-apoptotic proteins allow cells to evade apoptosis, a mechanism frequently defective in cancer affecting the response to therapy [5]. Thus, favoring apoptosis could represent a valuable approach to overcoming resistance.
In 2016, venetoclax/ABT-199, a specific Bcl-2 inhibitor, was approved by the FDA for the treatment of several kinds of leukemia and lymphoma, thus validating the relevance of targeting apoptotic machinery in onco-hematological neoplasms [6]. In addition, the first clinical study analyzing the efficacy of venetoclax in solid tumors evidenced a good activity in metastatic breast cancer [7]. Preclinical results led to the use of combinatorial regimes, including venetoclax or other inhibitors of the Bcl-2 family, in clinical trials under evaluation in cancers of different origins [8,9].
Experimental findings support the use of anti-apoptotic protein inhibitors in combination therapy both in melanoma and ovarian carcinoma. Inhibition of BRAF and/or MEK/ERK pathways, which together with immunotherapy represent the standard-of-care for melanoma treatment, has been reported to modulate the expression of some members of the Bcl-2 family [10][11][12][13][14][15][16][17][18][19][20][21]. Analysis of melanoma biopsies after treatment with BRAF inhibitors (BRAFi) further supported these preclinical observations, i.e., demonstrating lower levels of NOXA protein [20] and higher expression of BIM and BID proteins, as well as an inverse correlation between Bcl-2 protein expression and patient outcome [16]. Moreover, Bcl2A1/BFL1 anti-apoptotic protein expression in melanoma patients has been positively correlated with the response to BRAFi [22,23]. Pro-apoptotic and anti-apoptotic proteins have also been identified as critical mediators of the resistance to BRAFi and MEK inhibitors (MEKi) [15,17,[24][25][26]. Similarly, Bcl-2 family members play a role in the apoptosis induced by chemotherapy and in chemoresistance in ovarian cancer [27][28][29].

Cell viability and clonogenic ability
For cytotoxicity experiments, cell lines were plated in 96-well plates and, after 24 h, were treated with different drugs. MTT assay (Sigma-Aldrich) was used for melanoma, pancreatic, and NSCLC cells exposed to single agents, whereas CellTiter-Glo® Luminescent assay (Promega, Southampton, UK) was used for combinatorial regimes in melanoma. MTS (Promega) was used for ovarian cancer cells. For T-ALL, the number of viable cells was counted by a Trypan Blue exclusion assay (Sigma-Aldrich). The count was performed with hand tally counter and hemocytometer (Neubauer cell chamber). Loewe Scores were calculated by SynergyFinder 2.0 software [42]: scores <0, 0≤ scores >5, and ≥5 indicated, respectively, antagonistic, additive, and synergistic effects. GraphPad Prism software (Dotmatics, Bishop's Stortford, UK) was used to evaluate the concentration of the drug reducing 50% cell viability (IC 50 ).
To evaluate the cell colony-forming ability in melanoma cells, after treatment cells were seeded (200 cells) into 60-mm Petri dishes. After 10 days, colonies were stained (2% methylene blue in 95% ethanol) and counted. All cell aggregates with more than 50 cells were considered colonies, while those containing less than or equal to 50 cells were not considered colonies. The surviving fractions were calculated as the ratio of absolute survival of treated sample/survival of the control sample.
For colony assay in OVCAR 5, cells were seeded (250 cells/mL) in 6-well plates and, after 48 h, were treated with olaparib and IS21, and colonies were left to grow for 10 days, then stained (Gram's Crystal Violet solution, Merck) and quantified (QICAM 32-0030 C camera, QIMAGING, and Colony plus 2.0 program), by setting standardized parameters, considering a cell aggregate containing at least 50 cells (e.g. area min 0.0025, area max 20.0000, background correction, edges exclusion).

Immunoprecipitation and western blot analyses
For immunoprecipitation experiments, A375 cells treated for 48 h with vehicle or 20 μM IS21 were washed three times with PBS, harvested, and lysed in RIPA buffer (Santa Cruz Biotechnology, Texas, USA) in the presence of proteases and phosphatases inhibitors. Protein extract was obtained by centrifugation (15 min, 10 000 × g, 4°C) and protein concentrations were measured in duplicate using a BCA (Pierce, Thermo Fisher Scientific) protein assay kit. Protein extracts (1500 μg) were pre-cleared for 2 h at 4°C by adding 30 μl Dynabeads Protein G (Invitrogen, Thermo Fisher Scientific). The pre-cleared lysates were transferred to a new tube and incubated overnight at 4°C with Bcl-2 (cat 4223), Bcl-xL (cat 2764), or Mcl-1 (cat 94296) antibodies (1:100, Cell Signaling, Milan, IT) or with 2 μg rabbit IgG (BETHYL Laboratories, Waltham, MA), as control. 60 μl Dynabeads Protein G were washed three times, and then the lysates were added and incubated for 2 h at room temperature. The beads were washed (1 ml PBS, 0.02% Tween) five times, and then the immunoprecipitated proteins were eluted with 30 μl Laemmli sample buffer 2×, without reducing agent, and boiled (10 min, 70°C). Immunoprecipitated proteins were separated from the beads and 30 μl of water was added to each sample to a final volume of 60 μl. One-third of eluted samples (500 μg of starting materials) for each condition were separated by 12% precast SDS-PAGE gel (Bio-Rad, Hercules, CA, USA).

Cytofluorimetric analysis
To analyze cell cycle distribution, cells were fixed with ice-cold 70% ethanol for 24 h at 4°C. Next, cells were washed in PBS buffer and stained with 500 μl of PBS containing RNase A (100 μg/ml, Sigma-Aldrich) and Propidium iodide (50 μg/ml) for 30 min in the dark.
Tumor growth was monitored by bioluminescent imaging once a week. The signal was detected using the IVIS Spectrum CT (Perkin Elmer, Waltham, MA, USA) and analyzed using the Living Image software version 4.7.4. Mice were anesthetized, i.p. injected with 75 mg/kg D-luciferin, and imaged 10 minutes after injection. For ex vivo experiments, after sacrifice, lung, spleen, and liver were explanted, immersed in a solution containing D-luciferin for 10 minutes, and analyzed by bioluminescent imaging. Photon emission was measured in specific regions of interest (ROIs). Data were expressed as photon/second/cm2/steradian. The intensity of bioluminescence was color-coded for imaging purposes. ROIs were also used to evaluate the response evaluation criteria in solid tumors (RECIST) modified for animal experimentation [46] and defined as follows: partial response (PR) for tumor change ≤−50%, stable disease (SD) for −50%< tumor change >35%, progression disease (PD) for tumor change ≥35%.

Cellular thermal shift assay (CETSA)
To perform CETSA, A375 cells (8 × 10 5 ) were seeded into 6-well plates in complete medium. The day after, the cells were treated for 4 h with 20 or 40 µM IS21 or 0.1% (v/v) DMSO, as control. After washing, the cells were collected, suspended in PBS and 6 aliquots were subjected to a 3 min incubation at different temperatures between 30 and 55°C. Subsequently, the cells were lysed by RIPA buffer and centrifuged. The soluble proteins were separated by a 12% SDS-PAGE and Western blot analysis was carried. Densitometric analyses of the resulting bands were carried out using the ImageJ software as above reported. GAPDH was used as a normalizer. The experiments were performed two times.

Statistical analysis
In vitro experiments were replicated at least three times, unless otherwise indicated, and the data were expressed as average ± standard deviation (SD) or as average ± standard error of the mean (SEM). Group differences were analyzed with a two-sided paired or unpaired Student's t-test.
In vivo experiments were repeated twice. Differences between groups, analyzed with T test, Wilcox, or Mann-Withney, were considered statistically significant for p < 0.05.
Statistical analyses were performed with GraphPad Prism 9 software. Sample sizes were chosen based on preliminary results to ensure a power of 80% and an alpha level of 5%. No data or animals were excluded from the analyses.
IS21 reduced the viability of four T-ALL cell lines differently, with IC 50 values at 48 h ranging from 5.4 to 38.43 μM (Fig. 1A). We next evaluated the possible association between IS21 activity and the levels of different anti-and pro-apoptotic Bcl-2 family proteins. IC 50 values of IS21 failed to correlate significantly with endogenous levels of the analyzed proteins (Fig. 1B). Notably, JURKAT cells, expressing lower levels of Bcl-2 protein, were the most resistant to IS21 (Fig. 1A, B). PARP cleavage, indicative of apoptosis triggering, was induced upon IS21 treatment in LOUCY and TALL1 cells with high levels of Bcl-2 protein. At the same time, it was not detected in the JURKAT cells with low Bcl-2 levels (Fig. 1C).
Accordingly to published data [43], ABT-199, a Bcl-2-specific BH3 mimetic, strongly reduced the viability of TALL1, LOUCY, and MOLT-3 cells, failing to significantly suppress JURKAT cell viability ( Supplementary Fig. 1A). We next explored the cytotoxic effect of IS21 in combination with doxorubicin and vincristine, two of the main chemotherapy drugs used in the first-line treatment of T-ALL [51]. We focused on the JURKAT cell line, the most resistant cell line to IS21 (Fig. 1A, B), demonstrating that IS21 was able to synergistically potentiate the efficacy of both drugs (Fig. 1D), with Loewe scores of 35.9 and 39.6 for doxorubicin and vincristine respectively. Importantly, IS21 + doxorubicin or vincristine treatments significantly increased the percentage of apoptotic cells compared to single agents (Fig. 1E).
Moving on to solid tumors, a heterogeneous response was observed, with IS21 IC 50 values ranging from 9.1 μM to 27.1 μM for melanoma, from 10.1 μM to 81.7 μM for ovarian carcinoma, from 7.7 μM to 35.9 μM for NSCLC, and from 21.4 μM to 65 μM for pancreatic carcinoma ( Fig. 2A To examine whether IS21 activity was dependent on the presence of the pro-apoptotic proteins BAX and BAK, we tested the effect of IS21 in the proliferation of A375 melanoma cells silenced for BAX, BAK, or both proteins (Fig. 2C). Interestingly, BAX + BAK double silenced cells were significantly more resistant to IS21 compared with control cells, being IC 50 values 10.9 ± 2.4 μM and 33.3 ± 10 μM for control transfected and BAX + BAK silenced cells, respectively (Fig. 2D, E). On the contrary, BAX or BAK single silencing was not sufficient to significantly increase the IS21 IC 50 value (Fig. 2D, E).
We also evaluated the capability of IS21 to interact actually with the three anti-apoptotic proteins in the cell environment by CETSA [52]. To this purpose, A375 cells were treated for 4 h with IS21 (20 and 40 μM), exposed for 3 min to different temperatures (30-55°C), and then subjected to non-denaturing lysis to measure the residual amount of soluble Bcl-2, Bcl-xL, and Mcl-1 proteins by Western blot analysis. The results obtained, showed an evident stabilization of Bcl-2 protein at 45°C and, to a lesser extent, at 50°C in the samples incubated with 20 or 40 μM IS21, thus confirming the occurrence of an effective Bcl-2/IS21 interaction in the A375 cells ( Supplementary Fig. 2A). Conversely, we did not observe any effect of the IS21 treatment on Bcl-xL and Mcl-1 proteins thermal stability ( Supplementary Fig. 2B, C).
We next tested the effect of a panel of pan or specific Bcl-2 family inhibitors on the cell viability of melanoma and ovarian cancer cells. When melanoma (Fig. 3A) and ovarian cancer (Fig. 3B) cells were exposed to increasing concentrations of the pan-inhibitor, ABT-263, a strong reduction of cell proliferation was observed, even if higher heterogeneity was observed in melanoma (IC 50 (Fig. 3D, F, H, J), even with a different extent. In fact, melanoma (Fig. 3I) and ovarian cancer (Fig. 3J) cells were more resistant, respectively, to WEHI-539 and S63845 than to the other inhibitors tested.
BH3 mimetics synergistically potentiate the effect of PARP inhibition in ovarian cancer We next investigated the effect of Bcl-2 family inhibitors on the sensitivity of ovarian cancer cells to olaparib, a PARP inhibitor approved for the maintenance setting in ovarian cancer [53], that has been reported to alter the expression of Bcl-2 family proteins in ovarian cancer cell lines [38]. We focused on OVCAR 5, the most resistant cell line to IS21 (Figs. 2B, 3J) and among the most  Fig. 3A). The treatment with olaparib increased the expression of Bcl-2 protein, while it did not affect the expression of other Bcl-2 family proteins ( Supplementary Fig. 3B). Exposure to increasing concentrations of olaparib+IS21 combination elicited a stronger growth inhibitory effect than that obtained with single agents (Fig. 4A). Notably, the two compounds were synergistic in almost all the experimental points (Loewe score ≥5), with an overall Loewe score = 20.97 (Fig. 4B). Furthermore, the combined treatment significantly reduced the clonogenic ability when compared to single agents (Fig. 4C).

IS21 and ABT-199 synergistically potentiate the effect of MEK inhibition in BRAFwt melanoma cells
Since BRAF mutation drives the therapeutic decision in metastatic melanoma patients [54], we compared the IS21 and ABT-199 IC 50 values between BRAFwt and BRAF mutated melanoma cells ( Supplementary Fig. 4A), evidencing a not significantly difference between the two groups.
Next, we analyzed the effect of IS21 and ABT-199 combined with the MEKi, trametinib, in Sbcl1 BRAFwt melanoma, whose therapeutic options are much less than BRAF mutated ones [55]. Trametinib treatment in Sbcl1 strongly increased the levels of BIM protein, while it did not affect the expression of other Bcl-2 family proteins ( Supplementary Fig. 4B). The simultaneous exposure to IS21 and trametinib induced a more potent inhibition of cell proliferation compared to single treatments: a synergistic effect of the two drugs in every experimental point tested was observed, with an overall Loewe score of 21.31 (Fig. 5A, B). Furthermore, the combined treatment significantly reduced the clonogenic ability compared with single agents (Fig. 5C). Analysis of cell cycle distribution evidenced an increased percentage of cells in the subG1 peak, indicative of cell death, after combined treatment (Fig. 5D, Supplementary Fig. 5A), and addition of zVAD to the combination, strongly decreased the percentage of cells in the subG1 peak (Fig. 5D).
The combined treatment trametinib+ABT-199 produced a synergistic effect (Loewe score = 11.76) on growth inhibition (Fig. 5E) when Sbcl1 cells were treated with doses of ABT-199 lower than 25 µM (Fig. 5F). A significant reduction of clonogenic ability (Fig. 5G) paralleled by a significant increase of cells in the subG1 peak (Fig. 5H, Supplementary Fig. 5A), was observed after combined treatment compared with single agents. The addition of zVAD significantly reduced the percentage of cells in the subG1 peak (Fig. 5H).

IS21 and ABT-199 sensitize BRAF mutant melanoma cells to MAPKi in vitro
BRAFi, in combination with MEKi, represents the standard-ofcare for advanced BRAF-mutated melanoma [54]. In agreement with previously published papers demonstrating BIM Results represent the mean ± SD of three independent experiments. I, J p-values were calculated between cells treated with each compound to all the others, *p < 0.05, **p < 0.01, and ***p < 0.001. upregulation after BRAFi treatment [18,56], BRAFi altered the expression of Bcl-2 family proteins in A375 melanoma cells, as demonstrated by the reduction of Bcl-2 and the increase of BIM proteins ( Supplementary Fig. 3B). Importantly, the simultaneous treatment of IS21 and dabrafenib, a BRAFi, inhibited the proliferation of both A375 (Fig. 6A, Supplementary Fig. 4C) and M14 (Supplementary Fig. 4D, E) cells with a synergistic interaction (Loewe score = 12.6 for A375 and 16.2 for M14). The combination treatment also reduced the clonogenic ability of A375 cells compared with single compounds (Fig. 6B). A significant increase of cells in the subG1 peak was also observed: 45% after combined treatment compared with single treatment with dabrafenib (23%) or IS21 (15%) in A375 cells (Fig. 6C, Supplementary Fig. 5B) and of 30% in the combination compared with <10% of single agents in M14 cells (Supplementary Fig. 4F). The addition of zVAD significantly reduced the percentage of cells in the SubG1 peak in A375 cells treated with the combination (Fig. 6C). Enhanced apoptosis after the combined treatment was also confirmed by significant increase of annexin V and active caspase 3 positive cells when compared to single agents (Fig. 6D, Supplementary Fig. 5C, D).

IS21 and ABT-199 sensitize BRAF mutant melanoma cells to
MAPKi in vivo IS21 and ABT-199, administered as single agents, were previously reported to reduce in vivo tumor growth of melanoma models [41]. To evaluate their effect in combinatorial regimes and to validate our results in vivo, mice carrying A375luc tumors were treated with double (dabrafenib+trametinib) or triple (dabrafenib +trametinib+IS21 or dabrafenib+trametinib+ABT-199) combinations (Fig. 7A). Dabrafenib+trametinib caused an initial tumor shrinkage until day 20 when tumors started to regrowing (Fig. 7B, C, F). Notably, the addition of IS21 significantly reduced the tumor growth compared with dabrafenib+trametinib combination (Fig. 7B, C). Assessing the therapeutic response of single mice by using RECIST-like criteria, we found that 5 out of 6 mice treated with the triple combination reached partial response, compared to 2 out 6 in the dabrafenib+trametinib group (Fig. 7D). Similarly, the triple combination dabrafenib+trametinib+ABT-199  p-values were calculated between single and combination treatments, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, and between cells with or without zVAD, # p < 0.05 and ## p < 0.01. p-values were calculated between single and combined treatments, *p < 0.05; **p < 0.01; ***p < 0.001, and between cells with or without zVAD, # p < 0.05. significantly reduced the tumor growth compared with the double combination, achieving a longer disease control until the day of sacrifice (Fig. 7B, E), with 5 out of 6 mice responding to treatments (Fig. 7F). Importantly, the effect of the triple treatments, evaluated by the ex vivo imaging of organs, revealed a reduction, even though not significant, on the ability of A375 to colonize lung, spleen, and liver compared with dabrafenib+trametinib (Supplementary Fig. 6A, B). Of note, all treatments were highly tolerated and no significant changes in body weight ( Supplementary  Fig. 6C), diet consumption, and postural/behavioral habits were observed.
Here, we demonstrated that IS21 reduced the viability of cell lines derived from different tumor histotypes, confirming the relevance of the Bcl-2 family in T-ALL, melanoma, lung, pancreatic and ovarian carcinomas, and fostering their use in a broad spectrum of cancers.
We also identified Bcl-xL and Mcl-1 protein levels as markers of IS21 sensitivity in melanoma and ovarian cancer cell lines, respectively, and demonstrated that the anti-proliferative effect of IS21 was dependent on BAK and BAX expression.
We also deepened the action of IS21 from a mechanistic point of view. Our analysis of Bcl-2, Bcl-xL, and Mcl-1 immunoprecipitates demonstrated that IS21 induced a re-distribution of some pro-apoptotic proteins, strongly reducing the interaction of either Bcl-2 or Bcl-xL with their main binding partners (BAX and Beclin-1 for Bcl-2; BAX and BAK for Bcl-xL), but not that of Mcl-1. Thus, under the condition of IS21 plus target therapy, BAX and BAK could potentially be activated by BIM, which is increased by target therapy. Our results suggested that BAX and BAK, being the main interactors of Bcl-2 and Bcl-xL, were necessary to the IS21 activity, although their baseline levels did not predict the IS21 sensitivity in T-ALL nor in melanoma or ovarian cancer models.
Moreover, docking experiments indicated that IS21 was able to displace the BH3 helix more effectively from Bcl-2 and Bcl-xL than from Mcl-1. Using CETSA experiments, we confirmed the direct interaction of IS21 with Bcl-2 inside the cancer cell. The lack of interaction between the compound and Bcl-xL or Mcl-1 could depend on a lower affinity of IS21 for these proteins, as already suggested by SPR data [40]. However, it should be emphasized that in many cases the expression level of a protein, the localization, and physiological involvement in intracellular interactions can significantly influence its effective ability to bind a compound inside the cell [61]. In fact, it has been reported that the three proteins are differently expressed and localized in melanoma cells (https://www.proteinatlas.org/humanproteome/ pathology, accessed on July 13th, 2023) [62] and, therefore, possibly differently reachable from soluble BH3 inhibitors. Next, we demonstrated that a panel of pan or specific Bcl-2 family inhibitors was able to reduce the viability of different cancer cell lines, even if to a different extent, confirming the relevance of the Bcl-2 family also in solid tumors, and fostering their use in a wide spectrum of cancers. In melanoma, a specific Bcl-xL inhibitor was less effective than the others BH3 mimetics. Further analyses are needed to test other Bcl-xL inhibitors to confirm this observation.
In combination experiments, we demonstrated that IS21 sensitized T-ALL cells to the treatment with doxorubicin or vincristine, increasing apoptosis. Interestingly, a phase II clinical trial is recruiting T-ALL and lymphoblastic lymphoma patients to evaluate the efficacy of ABT-199 in combination with chemotherapy, including vincristine and doxorubicin (NCT00501826).
In ovarian cancer cell lines, the pan inhibitors IS21 and ABT-263 synergistically potentiated the effect of olaparib, whereas ABT-199 induced an additive effect. Published data demonstrated that targeting Bcl-xL, but not Bcl-2, sensitized ovarian cancer to PARP inhibition [38,39]. Higher levels of Bcl-xL protein compared to Bcl-2 and the olaparib-induced increase of Bcl-xL could explain why Bcl-2 specific inhibition was less effective in potentiating PARPi, when compared to pan BH3 mimetics [38]. Moreover, Dutta and colleagues identified a novel interaction between Bcl-2 and PARP1 that interfered with PARP1 activity, and demonstrated that Bcl-2 overexpression phenocopies PARP inhibition in chronic lymphocytic leukemia, breast and lung cancer [63]. We cannot exclude that this interaction can also occur in ovarian cancer, and could play a role in the sensitivity of the combination of PARPi with Bcl-2 specific inhibitors. Based on these preclinical findings, several clinical trials are ongoing to study the efficacy of BH3 mimetics alone or in combination in ovarian cancer, and a recent phase II study demonstrated that navitoclax showed an acceptable tolerance profile but poor activity in women with platinumresistant/refractory recurrent epithelial ovarian cancer [64]. A phase I study is recruiting women with recurrent high-grade serous carcinoma who have progressed after platinum-containing chemotherapy to evaluate the combination of olaparib and navitoclax (NCT05358639).
Our data also demonstrated that IS21 and ABT-199 reduced melanoma cell viability in a BRAF-independent manner, supporting the fact that both BRAFwt and BRAF mutated melanoma cells may benefit from the treatment with Bcl-2 family inhibitors.
In BRAFwt melanoma cells, the combination of ABT-199 or IS21 with trametinib was more active than single treatments in terms of reduction of cell proliferation and clonogenic ability, and induction of apoptosis. Apoptosis was rescued when zVAD was added to the combined treatment, confirming that BH3 mimetics increase the sensitivity to trametinib through the apoptotic pathway. The ability of some inhibitors of anti-apoptotic proteins to increase the sensitivity to target therapy in the BRAFwt/RAS mutated melanoma subgroup has also been reported. ABT-263 synergizes with MEK1/2i and induces death in melanoma cell lines with BRAF or RAS mutations [18]. These findings open the possibility of treating this specific subgroup of melanoma patients, who have limited access to active therapy, with BH3 mimetics. A phase I/II study is active to test the trametinib and navitoclax combination in KRAS or NRAS mutation-positive patients with metastatic/refractory/unresectable malignant solid neoplasms, including melanoma (NCT02079740).
We also found that ABT-199 and IS21 sensitized BRAF-mutated melanoma cells to dabrafenib and dabrafenib+trametinib treatments in terms of reduction of cell viability and clonogenic ability. Analysis of caspase 3 activation, annexin V positive cells, subG1 peak in the cell cycle distribution, and the use of caspase inhibitors demonstrated increased apoptosis after combined treatments. As we have previously demonstrated that IS21 could induce apoptosis through the alteration of autophagy in different tumors [41], we can hypothesize that the IS21 effect herein reported on cell viability/death may be mediated, at least in part, by the autophagic pathway.
Most notably, both ABT-199 and IS21 strongly potentiated the effect of dabrafenib+trametinib in vivo, by reducing tumor growth and metastatic ability and achieving longer disease control, with no evident signs of toxic effects. These data strongly support the clinical development of BH3 mimetic-based combinations.
The ability of specific or pan inhibitors of anti-apoptotic proteins to increase the efficacy of therapies in melanoma is not a new concept [8], but none of these studies, except the study of Rohrbeck's group [15], analyzed the effect of ABT-199. In particular, our results were in agreement with those demonstrating the ability of ABT-737 [30] and ABT-263 [31], to potentiate the efficacy of the BRAFi in BRAF-mutated melanoma models [11,13,15,16,18,19]. In vitro and in vivo ability of ABT-737 or TW-37 to synergize with MEKi has also been reported in melanoma models carrying BRAF mutation [17,26]. The inefficacy of ABT-199 to affect the sensitivity of BRAF mutated melanoma cells expressing Bcl-2 protein to BRAFi, demonstrated by Rohrbeck's group, could be due to different scheduling used [15]. More importantly, ABT-737 and ABT-263 and the Bcl-xL specific inhibitor, A1155, were also found to re-sensitize in vitro/in vivo melanoma models resistant to BRAFi or MEKi [11,15,16]. Recently, also Mcl-1 inhibitors showed their efficacy in potentiating the activity of BRAFi in melanoma models [19,20,32]. All this evidence supports the relevance of BH3 mimetics for melanoma treatment. To this purpose, a phase I/II study is active to evaluate the efficacy of navitoclax in combination with dabrafenib/trametinib in BRAF mutant melanoma patients (NCT01989585).
In conclusion, our findings suggest new therapeutic strategies active in poorly responsive melanoma and ovarian carcinoma models, both using agents already approved, i.e., venetoclax, with possible rapid clinical translatability, or agent under development, i.e. IS21. We know that IS21 is effective at micromolar concentrations, but it is a compound that can still be optimized before becoming a drug. In this context, we hypothesized chemical modifications (replacement of benzofurane moiety with other bicyclic heterocycles, insertion of methylene groups) to improve its efficacy.

DATA AVAILABILITY
Data and materials are available upon request to the corresponding author.